1 Department of Micro- and Nanotechnology, Technical University of Denmark2 Colloids and Biological Interfaces, Department of Micro- and Nanotechnology, Technical University of Denmark3 Department of Chemistry, Technical University of Denmark4 Physical and Biophysical Chemistry, Department of Chemistry, Technical University of Denmark

Abstract:

In spite of intensive research efforts over the past decades, the mechanisms by which membrane-active antimicrobial peptides interact with phospholipid membranes are not yet fully elucidated. New tools that can be used to characterize antimicrobial peptide-lipid membrane interactions are therefore highly warranted. Fluorescence correlation spectroscopy is a biophysical technique that can be used to quantify leakage of fluorescent probes of different sizes from large unilamellar vesicle, thereby potentially becoming such a new tool. However, the usage of fluorescence correlation spectroscopy to quantify leakage from large unilamellar vesicles is associated with a number of experimental pitfalls. Based on theoretical and experimental considerations, we discuss how to properly design experiments to avoid these pitfalls. Subsequently, we apply fluorescence correlation spectroscopy to quantify leakage of fluorescent probes of different sizes through transmembrane pores formed by each of the three representative antimicrobial peptides: melittin, magainin 2, and mastoparan X. The experimental results demonstrate that leakage assays based on fluorescence correlation spectroscopy offer new and detailed insight into the size and cooperative nature of transmembrane pores formed by antimicrobial peptides that is not available from the conventional quenching-based leakage assays.